Exposure of iPSC-derived human microglia to brain substrates enables the generation and manipulation of diverse transcriptional states in vitro

Microglia, the macrophages of the brain parenchyma, are key players in neurodegenerative diseases such as Alzheimer’s disease. These cells adopt distinct transcriptional subtypes known as states. Understanding state function, especially in human microglia, has been elusive owing to a lack of tools to model and manipulate these cells. Here, we developed a platform for modeling human microglia transcriptional states in vitro. We found that exposure of human stem-cell-differentiated microglia to synaptosomes, myelin debris, apoptotic neurons or synthetic amyloid-beta fibrils generated transcriptional diversity that mapped to gene signatures identified in human brain microglia, including disease-associated microglia, a state enriched in neurodegenerative diseases. Using a new lentiviral approach, we demonstrated that the transcription factor MITF drives a disease-associated transcriptional signature and a highly phagocytic state. Together, these tools enable the manipulation and functional interrogation of human microglial states in both homeostatic and disease-relevant contexts.

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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy All iMGL data is deposited on Terra, including raw and Cell Ranger output of iMGL (H1 and CW50118, CW500036 and CW70437) single cell RNA-sequencing, fastq and bam files of iMGL untreated and treated with apoptotic neurons for ATAC-seq and fastq and bam files of MITF overexpressing and mCherry control bulk RNAsequencing. Raw data is available via managed access at DUOS https://www.duos.org; ID: DUOS-000151. Any additional data and code is available from the corresponding authors. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Sample size
For single cell analysis , ATAC-sseq and bulk RNA-seq , sample size was determine based on standards in the field (see PMID: 35953545 for example) . For immunocytochemistry and in situ hybridization at least 50 cells were measured across 2 biological replicates for each conditions. For qRT-PCR, at least 4 biological replicates were done for each conditions. All experiments were done across multiple differentiations and all conditions were done on each differentiation. Biolegends and TREM2 antibodies were validated by the manufacturer and used previously in PMID: 28426964 Biolegend antibodies validation from manufacturers: To ensure they are both specific and sensitive, we validate our antibodies through a variety of methods including:Testing on multiple cell and tissue types with a variety of known expression levels.Validation in multiple applications as a cross-check for specificity and to provide additional clarity for researchers. Comparison to existing antibody clones.
Using cell treatments to modulate target expression, such as phosphatase treatment to ensure phospho-antibody specificity.
Cells snailing antibodies were previously validated by the manufacturer Cell signaling : To ensure our antibodies will work in your experiment, we adhere to the Hallmarks of Antibody Validation™, six complementary strategies that can be used to determine the functionality, specificity, and sensitivity of an antibody in any given assay. CST adapted the work by Uhlen, et. al., ("A Proposal for Validation of Antibodies." Nature Methods (2016)) to build the Hallmarks of Antibody Validation, based on our decades of experience as an antibody manufacturer and our dedication to reproducible science.

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Cell line source(s) The following iPSC lines used in Figure 6 and Figure S12 were obtained from the CIRM hPSC Repository funded by the Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation iMGLs were detached using cold PBS, then resuspended in FACS buffer (PBS containing 2%BSA and 0.05mM EDTA). Samples were incubated for 15 mins in human Fc block (BD Biosciences) followed by 1h staining with conjugated antibodies (see below) at 4C. Samples were washed 3x with FACS buffer and resuspended in 500ul of FACS buffer for flow cytometry Instrument CytoFLEX S analyzer (Beckman Coulter)

Software
All analyses were performed using FlowJo V10 and statistical analysis performed using Prism9.
Cell population abundance shown in each Figure S1A, S3B Gating strategy all flow cytometry experiments ( S1A-B, S3A) were gated based on cell vs debris ( FCS-A vs SSC-A), singlets (FSC-A vs FSC-H), live (DAPI negative), specific gatings for antibodies was based on negative unstained sample and for phagocytosis sample without phagocytosis substrate.
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